Etiologic Risk Group (see link below): Risk Group 2
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Mycobacterium Tuberculosis organisms are obligate aerobes growing most successfully in tissues abundant with oxygen, such as the lungs. They are considered as "facultative intracellular pathogens" and usually affect mononuclear phagocytes such as macrophages. Physically, they are hydrophobic and have a high lipid content in the cell wall. Because the cells are hydrophobic and tend to clump together, they are impervious to classic staining techniques (Gram's stain). The disease caused by the organism, Tuberculosis, generally includes an onset of symptoms containing fever, weight-loss, constant cough, and lethargy. Today TB is prevented in most developed countries via vaccination at birth. If an individual has not garnered immunity and is infected at a later point, drugs such as INH, rifampin, pyrazinamide, or ethmbutol may be used.
Essentiality of this protein: Essential.
"1-Deoxy-d-xylulose 5-phosphate reductoisomerase catalyzes the NADPH-dependent rearrangement and reduction of 1-deoxy-d-xylulose 5-phosphate to form 2-C-methyld-erythritol 4-phosphate, as the second step of the deoxyxylulose 5-phosphate/methylerythritol 4-phosphate pathway found in many bacteria and plants. The end product, isopentenyl diphosphate, is the precursor of various isoprenoids vital to all living organisms." (http://journals.iucr.org/d/issues/2006/07/00/be5057/be5057.pdf)
Complex of proteins?: No.
Druggable Target: Yes. Drugability Index Score = .5 (TDR)
*length of your protein in Amino Acids: 413 Amino Acids Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 42853.4 g Molar Extinction coefficient of your protein at 280 nm wavelength: 37470 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. *CDS Gene Sequence (paste as text only):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
*NCBI Gene # or RefSeq#: 887800
*Locus Tag: RV2870C
*Protein ID (NP or XP #) or Wolbachia#: NP_217386.2
*Organism (including strain): Mycobacteria Tuberculosis
Etiologic Risk Group (see link below): Risk Group 2
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Mycobacterium Tuberculosis organisms are obligate aerobes growing most successfully in tissues abundant with oxygen, such as the lungs. They are considered as "facultative intracellular pathogens" and usually affect mononuclear phagocytes such as macrophages. Physically, they are hydrophobic and have a high lipid content in the cell wall. Because the cells are hydrophobic and tend to clump together, they are impervious to classic staining techniques (Gram's stain). The disease caused by the organism, Tuberculosis, generally includes an onset of symptoms containing fever, weight-loss, constant cough, and lethargy. Today TB is prevented in most developed countries via vaccination at birth. If an individual has not garnered immunity and is infected at a later point, drugs such as INH, rifampin, pyrazinamide, or ethmbutol may be used.
Essentiality of this protein: Essential.
"1-Deoxy-d-xylulose 5-phosphate reductoisomerase catalyzes the NADPH-dependent rearrangement and reduction of 1-deoxy-d-xylulose 5-phosphate to form 2-C-methyld-erythritol 4-phosphate, as the second step of the deoxyxylulose 5-phosphate/methylerythritol 4-phosphate pathway found in many bacteria and plants. The end product, isopentenyl diphosphate, is the precursor of various isoprenoids vital to all living organisms." (http://journals.iucr.org/d/issues/2006/07/00/be5057/be5057.pdf)
Complex of proteins?: No.
Druggable Target: Yes. Drugability Index Score = .5 (TDR)
*EC#: 1.1.1.267
Link to BRENDA EC# page:
1. http://www.brenda-enzymes.org/literature/lit.php4?e=1.1.1.267&r=654748
2. http://www.brenda-enzymes.org/literature/lit.php4?e=1.1.1.267&r=671075
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Spectrophotometric Assay (NADPH Absorption)
-- link to Sigma (or other company) page for assay or assay reagents (substrates): http://www.eenzyme.com/elitenadpnadphassaykitredfluorescence.aspx
-- link (or citation) to paper that contains assay information: http://journals.iucr.org/d/issues/2006/07/00/be5057/be5057.pdf
-- List cost and quantity of substrate reagents and supplier: $379/kit. 400 assays in 96-well plates.
Structure Available (PDB or Homology model): Yes, PDB. PDB ID: 2Y1E
---- Query Coverage: N/A
---- Max % Identities: N/A
---- % Positives: N/A
---- Chain used for homology: N/A
Current Inhibitors: 33 Current Inhibitors
List: http://www.bindingdb.org/jsp/dbsearch/PrimarySearch_ki.jsp?tag=pol&submit=Search&target=1-deoxy-d-xylulose%205-phosphate%20reductoisomerase&polymerid=50004227,50006037,4735
Expression Information (has it been expressed in bacterial cells): Yes.
Possible Transmembrane Helices
The sequence positions in brackets denominate the core region.
Only scores above 500 are considered significant.
Inside to outside helices : 6 found
from to score center
13 ( 15) 32 ( 32) 409 23
99 ( 99) 116 ( 116) 491 107
246 ( 246) 264 ( 264) 74 256
311 ( 311) 329 ( 329) 215 319
337 ( 337) 357 ( 355) 963 345
385 ( 385) 404 ( 402) 540 394
Outside to inside helices : 5 found
from to score center
15 ( 15) 32 ( 32) 243 23
99 ( 99) 117 ( 117) 616 109
311 ( 311) 329 ( 329) 345 319
336 ( 336) 352 ( 352) 1020 344
382 ( 385) 403 ( 403) 230 395
Table of Correspondences
Helices shown in brackets are considered insignificant.
A "+"-symbol indicates a preference of this orientation.
A "++"-symbol indicates a strong preference of this orientation.
inside->outside | outside->inside
( 13- 32 (20) 409 + ) |( 15- 32 (18) 243 )
( 99- 116 (18) 491 ) | 99- 117 (19) 616 +
( 246- 264 (19) 74 ++ ) |
( 311- 329 (19) 215 ) |( 311- 329 (19) 345 + )
337- 357 (21) 963 | 336- 352 (17) 1020
385- 404 (20) 540 ++ |( 382- 403 (22) 230 )
Purification Method: Column chromatography. (http://pubs.acs.org/doi/pdf/10.1021/bi049974k)
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
1 mtnstdgrad grlrvvvlgs tgsigtqalq viadnpdrfe vvglaaggah ldtllrqraq
61 tgvtniavad ehaaqrvgdi pyhgsdaatr lveqteadvv lnalvgalgl rptlaalktg
121 arlalankes lvaggslvlr aarpgqivpv dsehsalaqc lrggtpdeva klvltasggp
181 frgwsaadle hvtpeqagah ptwsmgpmnt lnsaslvnkg leviethllf gipydridvv
241 vhpqsiihsm vtfidgstia qasppdmklp islalgwprr vsgaaaacdf htasswefep
301 ldtdvfpave larqagvagg cmtavynaan eeaaaaflag rigfpaivgi iadvlhaadq
361 wavepatvdd vldaqrware raqravsgma svaiastakp gaagrhastl ers
*length of your protein in Amino Acids: 413 Amino Acids
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 42853.4 g
Molar Extinction coefficient of your protein at 280 nm wavelength: 37470
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
ATGACTAACAGTACAGACGGTAGAGCTGATGGACGGTTGAGGGTAGTGGTTCTGGGGTCTACCGGGAGCATCGGT
ACACAGGCCTTGCAAGTCATCGCAGACAATCCTGACCGATTCGAGGTGGTAGGCTTGGCAGCGGGTGGAGCGCAC
CTAGATACACTCCTGCGGCAAAGGGCTCAGACTGGAGTTACGAATATCGCTGTCGCGGACGAACACGCCGCACAG
AGGGTAGGGGACATCCCGTATCACGGCAGCGACGCTGCCACACGTCTGGTGGAGCAGACGGAAGCCGACGTCGT
CCTCAACGCTTTAGTTGGTGCTCTAGGGCTCCGTCCAACGTTAGCTGCGCTAAAAACCGGCGCTCGTCTTGCATTAG
CTAACAAGGAGTCCCTGGTAGCAGGTGGAAGCTTAGTACTCAGGGCCGCAAGACCTGGACAGATTGTACCGGTCG
ATTCTGAGCATAGTGCACTCGCACAATGCCTACGCGGTGGAACCCCGGATGAAGTAGCGAAACTGGTATTAACTGC
CAGTGGCGGGCCATTCAGAGGATGGTCCGCCGCTGACTTAGAACATGTGACACCAGAACAGGCCGGAGCTCATCC
AACCTGGTCCATGGGCCCCATGAACACCCTGAATTCTGCAAGTTTGGTTAACAAAGGTTTAGAGGTGATAGAAACCC
ACCTCTTATTCGGGATCCCTTACGACCGAATTGATGTAGTGGTACACCCTCAGTCCATAATACATTCGATGGTCACGT
TCATCGATGGAAGTACTATCGCGCAGGCTTCACCACCGGATATGAAACTCCCGATATCTCTTGCGCTAGGATGGCCA
CGTCGGGTGTCGGGCGCCGCGGCGGCTTGCGACTTCCACACAGCATCTTCCTGGGAATTTGAGCCGCTTGACACTG
ACGTATCCCAGCCGTAGAGCTGGCACGTCAAGCGGGTGTTGCTGGGGGGTGCATGACCGCCGTATACAACGCTGC
TAACGAAGAGGCTGCTGCCGCATTTTTAGCTGGGAGAATAGGCTTCCCCGCCATAGTCGGAATAATTGCTGATGTGC
TCCATGCAGCCGATCAGTGGGCTGTCGAACCCGCGACGGTCGACGACGTCCTCGACGCACAACGTTGGGCCCGAG
AGCGCGCACAGAGAGCAGTTAGCGGTATGGCGAGTGTAGCTATTGCGTCAACGGCAAAGCCGGGCGCTGCCGGTA
GGCACGCCAGCACCCTAGAAAGATCA
*GC% Content for gene: 56.7% (http://www.sciencebuddies.org/science-fair-projects/project_ideas/Genom_GC_Calculator.shtml)
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):